FORENSIC SEROLOGYSEROLOGY - the analysis and study of blood and other bodily fluids in or for evidence in crimes - term used to describe a broad range of laboratory tests using reactions of blood serum and body fluids The serology section of a forensic laboratory may deal with any or all of the following: • blood typing • characterization of unknown blood • stain patterns for crime reconstruction • paternity testing • semen identification in rape cases • DNA techniques used for identification FORENSIC SEROLOGISTS – scientists who examine physical evidence with the intent of finding, identifying and individualizing stains of biological origin IMPORTANCE OF THE STUDY OF BLOOD 1. circumstantial or corroborative evidence against or in favor of a suspect/perpetrator; 2. evidence on disputed parentage; 3. evidence in the determination of length of death; 4. evidence in the determination of the direction of victim or suspect; and 5. evidence in the approximation of time of crime or death. BLOOD - circulating tissue of the body - complex mixture of water (plasma 55%), cells, enzymes, proteins, glucose, hormones, organic and inorganic substances - opaque, deep red color, slightly alkaline with pH of 7.35-7.45 - primarily functions to circulate and supply nutrients and oxygen to the entire body and to remove wastes HEMOGLOBIN – iron‐containing protein and binds about 97% of all oxygen in the body responsible for the RED COLORING of blood Blood Volumes in the Body 4-6L of blood in the body About 58% ‐Veins About 13% ‐Arteries About 12% ‐Pulmonary Vessels (Lungs) About 9% ‐Heart About 8% ‐Arterioles/Capillaries SECRETORS vs. NON-SECRETORS Secretors – have A and B antigens in non-blood fluid - 80% of population Remaining 20% of population 3 BLOOD CELLS 1.Non-secretors .Not useful for DNA analysis 6-8 μm in size. along with other substances. etc. biconcave discs (circular with flat edges) contains hemoglobin carries/transport O2 to different parts of the body possess chemical structures on their surfaces called antigens or agglutinogens About 45% total volume of blood Most abundant cell in the blood 2. LEUKOCYTES (WHITE BLOOD CELLS) responsible for the production/release of antibodies important to human immune system Produced in the bone marrows have a nucleus .made of 90% water and 10% solids which are largely proteins a.yellowish fluid or liquid in which the cells are suspended . lipids and iron c. ERYTHROCYTES (RED BLOOD CELLS) Have no nucleus . globulins – carries drugs. THROMBOCYTES (PLATELETS) Irregularly‐shaped. sex hormones. albumen – most abundant blood protein b. . semen. thyroid hormones. colorless bodies produced in the bone marrow Their sticky surface lets them.don’t have blood type antigens in saliva.a molecule that stimulates an immune response . fibrogen – produces FIBRIN responsible for hemostasis ANTIGENS OR AGGLUTINOGENS . form clots to stop bleeding Only active when damage occurs to the circulatory system walls HEMOSTASIS – blood clotting HEMOLYSIS – blood agglutination or clumping BLOOD PLASMA (MEGAKARYOCYTES) .Useful for DNA analysis clean up dead cells and tissue debris that would otherwise accumulate and lead to problems 3.substances that induce a specific immune response and subsequently react with the products of a specific immune response .they stimulate antibody generations WALTER MORGAN AND WINIFRED WATKINS – discovered in 1953 the chemical structures of the 3 antigens A antigen – lipid tail – glu – gal – nag – gal (-nag) – fru B antigen – lipid tail – glu – gal – nag – gal (-ga) – fru O antigen – lipid tail – glu – gal – nag – gal – fru ***nag – N-acetylglucosamine . ANTIBODIES OR AGGLUTININS . Antibody A will cause A and AB blood to clot . proteins that are present in the serum . Antibodies produced by the A alleles remove any red blood cells containing B antigens by clumping them together . Type O . Type AB . Type B 3. Blood typing is done by reacting whole blood with anti-sera (anti-A and anti-B) .regarded as the universal recipient 4. Antibodies produced by the B alleles remove any red blood cells possessing A antigens 4 BLOOD TYPES 1. responsible for ensuring that the only blood cells that can survive in a person are cells of the correct blood type . Type A 2.regarded as the universal donor BLOOD TYPING . Antibody B will cause B and AB blood to clot . Type O blood contains not antigens so will not clot Blood Antigens on Red cells Antibodies in Serum Population type Frequency A A Anti-B 40-42% B B Anti-A 1012% AB AB Neither anti-A or anti-B 3-5% O Neither anti-A or Both anti-A and anti-B 43-45% anti-B . often referred to as the D antigen or RhD antigen .com/EBchecked/topic/500915/Rh-blood-group-system xxxx PROBABILITIES USING Rh BLOOD SYSTEM A+ 1 in every 3 AB+ 1 in 29 A. The disease can be avoided by vaccinating the mother with Rh immunoglobulin after delivery of her firstborn if there is Rh-incompatibility. Rh. B. Rh BLOOD SYSTEM .first system to be developed and recognized . etc…. O B B. Xg. ABO BLOOD SYSTEM . Treatment of erythroblastosis fetalis usually entails one or more exchange transfusions. or hemolytic disease of the newborn. Duffy. Cartwright. a small amount of the fetus’s blood may enter the mother’s bloodstream. 1 in 67 O.britannica. Lewis. Blood type Donor Recipient A A. which will attack any Rh- incompatible fetus in subsequent pregnancies.(absence of D antigen) while Rh+ (presence of D antigen) NOTE: Hemolytic Disease of the Newborn described by Levine and Stetson A similar hazard exists during pregnancy for the Rh-positive offspring of Rh-incompatible parents. which they named anti-R (1940) . WIENER injected animals with Rhesus monkey cells to produce an antibody which reacted with 85% of human red cells. 1 in 16 AB. the International Society of Blood Transfusion (ISBT) currently (2010) recognizes 26 blood group systems . The first child of such parents is usually in no danger unless the mother has acquired anti-Rh antibodies by virtue of incompatible blood transfusion. O O BLOOD TYPING SYSTEMS . AB. Scianna. Lutheran. O AB AB A. Diego. Kidd. O O A. 1 in 167 B+ 1 in 12 O+ 1 in 3 B. 1 in 15 .developed by the Austrian-American physician KARL LANDSTEINER in 1901 with his assistant Russian-American physician PHILIP LEVINE 2. AB. was first observed and developed from RHESUS MONKEYS . xxxxxhttp://www. MNS. AB A. Kell. During labour. KARL LANDSTEINER AND ALEXANDER S. which can be fatal to the fetus or to the infant shortly after birth. however. The mother will then produce anti-Rh antibodies. AB B. The Rh vaccine destroys any fetal blood cells before the mother’s immune system can develop antibodies. B. MOST COMMON BLOOD SYSTEMS: 1. This process produces erythroblastosis fetalis. when the mother is Rh-negative and the father is Rh-positive. swab the rectal canal. PHYSICAL EVIDENCES TO BE COLLECTED 1. RECTAL SWABS AND SMEAR – to be taken when warranted by history. Immediate collection is required of all positively identified blood and suspected specimens. TRANSPORT OF BLOOD EVIDENCE/SAMPLES 1. Use separate paper one for each hand. Always remember that blood has a very little resistance to contamination and decomposition. 5. 9. 4. Using 2 additional swabs repeat swabbing and smear the swabs onto separate microscope slides allowing them to air dry before packing. 6. 6. PUBIC HAIR STANDARD OR REFERENCE – cut 15-20 full length hairs from the pubic area or skin line 3. 3. PACKAGING. 4. right side. VAGINAL SWABS AND SMEAR – using 2 swabs simultaneously. ORAL SWABS AND SMEAR – to be taken if oral-genital happened. WHAT IS THE ANIMAL SPECIES OR WHAT IS THE HUMAN BLOOD TYPE? CHRONOLOGY OF BLOOD TESTING 1. smearing 1 of the swabs onto microscope slide.NOTE: BLOOD TYPE HEREDITY COLLECTION. BLOOD SAMPLE – collect a minimum of 7ml in a vacuum tube containing the preservative EDTA. URINE SPECIMEN – 30ml or more should be collected GENERAL PROTOCOLS FOR BLOODSTAIN CHARACTERIZATION 1. left side. 2. PRECIPITIN TEST 4. Fresh blood samples are the most valued. NaF can preserve it for a week at room temperature. FINGERNAIL SCRAPINGS – scrape the undersurface of the nails with a dull object over a piece of clean paper to collect debris. 8. Can be preserved infinitely between 40-50 degrees Celsius. 5. use 1 swab for smear and air dry before packing. IS IT HUMAN OR ANIMAL? 3. BLOOD GROUP TESTS 5. 7. DNA TESTS . Use 2 swabs simultaneously to swab the buccal area and gum line. Air dry before packing. A total of 50 hairs be cut and submitted. carefully swab the vaginal area and let the swabs air dry before packing. front. CONFIRMATORY TESTS 3. HEAD HAIRS – cut at skin line a minimum of 5 full length hairs from each of the following scalp locations: center. Using 2 swabs simultaneously. PRESUMPTIVE TESTS 2. IS IT BLOOD? YES or NO 2. PUBIC COMBINGS – place a paper towel under the buttocks and comb pubic area for loose or foreign hairs 2. back. Labeling and appearance of samples should be well noted. I.1g of luminol in 100ml of distilled water c. add a drop of 3% H2O2 POSITIVE RESULT: intense blue – bluish green FALSE POSITIVE: sputum. milk. 1-4-phthtalozinedione (luminol) b. Zn dust POSITIVE RESULT: deep pink FALSE POSITIVE: Cu salts. LEUCOMALACHITE GREEN TEST REAGENTS: a. benzidine solution (benzidine powder dissolved in glacial acetic acid) b. bile. BENZIDINE TEST REAGENTS: a. place small fragment of the stained material on a filter paper b. PRESUMPTIVE TESTS: A. Phenolphthalein. leucomalachite green solution (1g of leucomalachite dissolved I 48ml of glacial acetic acid diluted to 250ml water) b. cheese D. CONFIRMATORY TESTS A. GUAIACUM TEST REAGENTS: a. moisten strip and dip into sample RESULT: Greenish – intensity = concentration F. iron salts. LUMINOL TEST REAGENT: a. milk. HEMASTIX TEST/KIT– Bayer Corporation . 3% solution of H2O2 POSITIVE RESULT: bluish green FALSE POSITIVE: saliva. cheese C. KASTLE-MEYER TEST (PHENOLPHTHALEIN) REAGENTS: a. clay B. formalin. before use. add 0. dissolve 5g of NaCO3 and 0. 5-amino-2. fresh guaiac resin with 95% alcohol b.commercially prepared strips of cellulose that contain mixtures of 0-toluidine (2- methylaniline) and H2O2 PROCEDURE: a. gum. iron salts. TAKAYAMA TEST PROCEDURE: addition of PYRIDINE to blood RESULT: salmon pink crystals . plant juices.7g of sodium perborate to the sodium carbonate RESULT: release of photon (light) II. potatoes. KOH. pus. 3% solution of H2O2 or few drops of turpentine POSITIVE RESULT: intense blue FALSE POSITIVE: saliva. pus. nasal secretion. horse radish E. bile. add a drop of the benzidine solution c. rust. 3% solution of H2O2 PROCEDURES: a.3-dihydro. pus. rust. TEICHMANN TEST REAGENT: mixture of glacial acetic acid and NaCl RESULT: brownish crystals of pure hemin with violet to almost black sheen III. offender. Drip patterns 3. Cast-off Stains 2. BLOODTYPING BLOOD STAIN PATTERNS: Information Obtained: Origin of bloodstains Distance between point of impact and origin Type and direction of impact Object/weapon used Minimum number of blows Position of victim. GEL DIFFUSION – diffusion of antigens and antibodies 2.B. PRECIPITIN TEST OR OUCHTERLONY TEST 1. Swipe 4. ELECTROPHORESIS 3. Wipe (1) (2) (3) (4) . and objects Movement by victim or offender at scene Support/contradict witness statements Indicate staged/secondary scenes BLOODSTAIN PATTERNS: 1. Rougher surface = greater distortion IMPACT ANGLES . Horizontal drop creates circular pattern 2.Determine direction of travel CALCULATING IMPACT ANGLE .EFFECTS OF SURFACE TEXTURE 1.Greater angle = greater elongation .Use calculator or standard curve .Determine L/W ratio .Defined as the internal angle at which blood strikes a target surface . Forces of surface tension 3.Determine W/L ratio . THE POINTED END OF THE BLOODSTAIN ALWAYS FACES THE DIRECTION OF THE BLOOD’S SOURCE .POINT OF ORIGIN DIRECTION OF BLOODSTAIN . Bloodstains are usually not exactly ellipsoidal in shape. but they tend to have rounded edge at one end of the major axis and a pointed edge at the opposite end of the major axis . 2. Swabs should be taken on sterile gauze / cloth and their smears prepared on sterile slides. METHOD OF COLLECTION Handling of articles bearing stains should be done very c arefully to avoid damage to spermatozoa. METHODS APPLIED FOR DETECTION OF SEMINAL STAINS: 1. Incest (Sexual intercourse in blood relation) and Sexual Murders. BODY (MIDPIECE) 3. Seminal Plasma (90%) 3. Pillow cover.5-5ml of seminal fluid per ejaculation 1ml = 100M to 150M of spermatozoa 3 MAIN PARTS OF A PSERMATOZOON: 1. WHERE TO LOOK FOR SEMINAL STAINS 1. Scene of crime: On the floor or grass etc. 1.In case of false Accusation by a women . Carpet.). HEAD 2. Spermatozoa (10%) 2. Towel. FORENSICS OF SEMEN SEMEN . 3. 3. Vagina & pubic hair. TAIL DE FI N IT IO N O F TE R MS Aspermia : N o sem en eja c ula t ed Hema tos per mia : Bl o od pre se nt i n seme n Leuc ocy tos per mi a : Wh ite bl oo d ce lls pres e nt i n se me n Azospermia : N o spe r ma to zoa f o u nd i n seme n Oli gos per mia : Lo w s perm c o n ce ntra tio n Necr os per mi a : No l iv e s perm i n sem en MEDICOLEGAL SIGNIFICANCE OF DETECTION OF SPERM & SEMEN Rape. emitted from the urethra (tube in the penis) on ejaculation Semen contents: 1. Bestiality (Sexual intercourse by a human being with a lower animal like dogs. Body Perineum. Vaginal / anal / penile swabs should be sent along with their smears on slides. calves. Sodomy (Anal intercourse).viscid mucilaginous fluid with faint yellow or white or grey color. Samples should be dried in air at room t emperature (37°C) and swabs dispatched in sterile test tube and slides in clean wrappers. Clothes: Underwear. Epithelial Cell (< 1%) 2. Bed sheet. Physical Examination . thigh. 2. sheep etc. Non -specifics & false negative results are common. Ammonium Molybdate Test (Phosphorus) 8. the SEMA® assay. PROCEDURE: A few drops of watery solution of the stain is extracted and taken on a slide and a drop of Florence reagent (8%) W/V solution of Iodine in water containing 5% W/V of Potassium Iodide) is poured & allowed to mix slowly under a cover slip.9) and 1% W/V aqueous solution of disodium Phenyl phosphate are added. Chemical Examination 3. like non -specificity and lack of reproducibility. Florence Test 2. which. Microscopic Examination PHYSICAL EXAMINATIONS : Include Visual Examination. if absorbent. Barberio Test 3. BERBERIO’S TEST: Basis: Detection of Spermine Procedure: A few drops of Berberio’s reagent when added to spermatic fluid produces crystals of sperm in picrate (needle shaped. LDM Isoenzyme Method 5. rhombic & of yellow colour). On good substrata seminal stains may appear to be fluorescent under ultraviolet light. After 10 minutes the phenol is detect ed by the addition of 2 drops of phenol reagent & 2 drops of 20% W/V solution of sodium carbonate. PROCEDURE: The fluid obtained af ter thorough maceration of a small cloth piece (about 4mm 2 ) is placed in a cavity on a porcelain tile land two drops each of citrate buffer (Ph 4. Creatinine in Phosphokinase 7. Enzyme -linked immunosorbent assay (ELISA). generally needle shaped. NOTE: For various valid reasons. Acid Phosphatase Isoenzyme Test 6. ACID PHOSPHATASE SPOT TEST : Modified Fishman and Lerner’s method.2. CHEMICAL EXAMINATIONS: The tests used to detect Seminal Stains are: 1. Semen Specific Glycoprotein (P30 ) Test 9. . also acquire stiffness due to dried se men. for a seminal vesicle -specific antigen (SVSA) FLORENCE TEST Basis: Choline is detected in this method. formed with a few minutes. To naked eye seminal stains generally appear translucent or opaque spots. at times with yellowish tint and darker border depending on color and thickness of substrata. RESULT: Dark brown crystals of choline periodide. Acid Phosphatase Test 4. the Florence and Berberio’s tests have not been accepted universally. 99% water . Nasal Secretion. This method gives a specific biochemical detection of spermatozoa in semen in the presence of Vagtinal Fluid. Residue obtained is stained haematoxylin and eosin. After sonication for 5 minut es the extracts and the cloth pieces are transferred to separate micro scope slides and cloth pieces delicately teased with a needle. MICROSCOPIC EXAMINATION: The Microscopic detection of the seminal stains is based in morphology of spermatozoa. oligospermic azoospermic & vasectemized individuals. The method is sufficiently specific & applicable to semen derived from normal. With this method it is possible to detect both intact spermatozoa as well as the disconnected heads. 3. Threads are removed and the residual liquid is gently evaporated to dryness. Positive results are obtained in large number of cases. ACID PHOSPHATASE ISOENZ YME METHOD Procedure: Seminal stains are extracted with water and is used in polyacrylmile gel Electrphoretic method followed by staining with methyl belliferyl.RESULT: Blue colour developed which indicates the presence of acid phosphatase.5 ml of 0. Electrophoresis is carried out in refrigerators for 150 minutes using a current of 5 mA Isoenzyme bands are revealed by staining. Phosphate reagent enable the seminal acid phosphatase to be distinguished from that of other substance like vaginal secretions.1 ml of this mixture is subjected to vertical polyacryl amide gel electrophoresis. PROCEDURES: 1. 2. 3. Advantages: 1. 2. Isoenzyme pattern of human is different from that of animal.25 ml of clear extract is mixed with 0. 0. 4.25 ml of 40% W/V of sucrose. Can differentiate from vaginal secretions on pattern of bands.01 N HCL in small test tubes placed in a beaker containing water. 0. Blood. LDH ISOENZYME METHOD : Detection of Spermatozoa Procedure: Seminal stains are extracted with 1 ml of water. LDH isoenzyme is stable in stains for over 4 week. FORENSIC CHARACTERIZATION OF SALIVA Contents of Saliva: 1. Cloth pieces from different stains are taken in 0. Saliva & Urine. Fluorescence Microscopy: It is based on the principle that Y chromosome is fluorescent to quinacrine. factsonfile. 2004. Aligarh. pdf http://faculty. Mucin (protein helps in swallowing) 3.com/ Saferstein R. Biological Science Screening Workshop. Pearson Education South Asia Pte. TESTS FOR SALIVA: 1. AMU.com/EBchecked/topic/500915/Rh-blood-group-system . Benedict’s Test 2. CRIMINALISTICS: An Introduction to Forensic Science. Seminar: Detection of Semen & Seminal Fluid Stains. Sabri I. Molisch Test REFERENCES: Newton DE. http://www. 8th Ed.htm http://www. 2007. Cheek cells / squamos cells (good for DNA) Adults produce 1. Amylase (enzyme to help digest carbohydrates) 4.5 liters of saliva/day and it is not uncommon at crime scenes (especially involving bite marks). Forensic Chemistry. nfstc – science serving justice.JN Medical College. Ltd.edu/mljensen/BloodBank/lectures/RhBloodGroupSystem. Department of Forensic Medicine and Toxicology. 2. Philippines.britannica.0-1.matcmadison.